JPET Fast Forward. Published on October 31, 2005 as DOI: 10.1124/jpet.105.093831 JPET FastThis articleForward. has not Published been copyedited on and October formatted. 31,The final2005 version as DOI:10.1124/jpet.105.093831 may differ from this version. JPET# 93831 Blood glucose lowering nuclear receptor agonists only partially normalize hepatic gene expression in db/db mice Downloaded from jpet.aspetjournals.org Michael Loffler, Martin Bilban, Mark Reimers, Werner Waldhäusl and Thomas M. Stulnig at ASPET Journals on September 25, 2021 Clinical Division of Endocrinology and Metabolism, Department of Internal Medicine III, Medical University Vienna, Vienna, Austria (M.L., W.W., T.M.S.); CeMM - Center of Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria (M.L., W.W., T.M.S.); Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University Vienna and Ludwig Boltzmann Institute for clinical and experimental Oncology, Vienna, Austria (M.B.); Laboratory of Molecular Pharmacology, National Cancer Institute, Bethesda, Maryland (M.R.) 1 Copyright 2005 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on October 31, 2005 as DOI: 10.1124/jpet.105.093831 This article has not been copyedited and formatted. The final version may differ from this version. JPET# 93831 Running Title Blood Glucose Lowering Drugs and Hepatic Gene Expression Corresponding author Downloaded from Thomas M. Stulnig, Clinical Division of Endocrinology and Metabolism, Department of Internal Medicine III, Medical University Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria; e-mail : [email protected] jpet.aspetjournals.org Number of Text pages 23 at ASPET Journals on September 25, 2021 Tables 2 Figures 6 + 3 Supplementary Figures References 40 Number of words Abstract 220 Introduction 333 Discussion 402 ABBREVIATIONS PPAR, peroxisome proliferator-activated receptor; QPCR, quantitative real-time reverse-transcriptase PCR; SREBP, sterol regulatory element-binding protein; UPR, unfolded protein response. Section Endocrine and Diabetes 2 JPET Fast Forward. Published on October 31, 2005 as DOI: 10.1124/jpet.105.093831 This article has not been copyedited and formatted. The final version may differ from this version. JPET# 93831 ABSTRACT Agonists of the nuclear receptors peroxisome proliferator-activated receptor (PPAR) γ, PPARα, and liver X receptors (LXR) reduce blood glucose in type 2 diabetic patients and comparable mouse models. Since the capacity of these drugs to normalize hepatic gene expression is not known we compared groups of obese diabetic db/db mice treated with agonists for PPARγ (rosiglitazone [Rosi]; 10 mg/kg/day), PPARα (Wy 14643 [Wy]; 30 mg/kg/day), and LXR (T0901317 [T09]; 40 Downloaded from mg/kg/day) and from untreated non-diabetic littermates (db/+) by oligonucleotide microarrays and quantitative reverse transcriptase polymerase chain reaction. The jpet.aspetjournals.org 10-day treatment period of db/db mice with Rosi, Wy, and T09 altered expression of 300, 620, and 735 genes including agonist-specific target genes, respectively. However, from the 337 genes differentially regulated in untreated db/+ vs db/db at ASPET Journals on September 25, 2021 animals, only 34 (10%), 51 (15%) and 82 (24%) were regulated in the direction of the db/+ group by Rosi, Wy and T09, respectively. Gene expression normalization by drug treatment involved glucose homeostasis, lipid homeostasis and local glucocorticoid activation. In addition, our data pointed to hitherto unknown interference of these nuclear receptors with growth hormone receptor gene expression and endoplasmic reticulum stress. However, many diabetes-associated gene alterations remained unaffected or were even aggravated by nuclear receptor agonist treatment. These results suggest that diabetes-induced gene expression is minimally reversed by potent blood glucose lowering nuclear receptor agonists. 3 JPET Fast Forward. Published on October 31, 2005 as DOI: 10.1124/jpet.105.093831 This article has not been copyedited and formatted. The final version may differ from this version. JPET# 93831 INTRODUCTION Modern anti-diabetic agents targeting nuclear receptors are designed to modulate blood glucose levels and gene expression on a molecular level. Transcriptional regulation by agonists of peroxisome proliferator-activated receptor (PPAR)γ, PPARα and LXR comprise genes involved in gluconeogenesis, fatty acid metabolism and ketogenesis (Willson et al., 2000; Venkateswaran et al., 2000). PPARγ is a critical transcription factor involved in energy Downloaded from balance and activated by well-established anti-diabetic drugs, the thiazolidinediones (Lehmann et al., 1995). Nuclear receptor agonist or fatty acid dependent activation of PPARα promotes peroxisomal proliferation, hepatic fatty acid oxidation and the generation of ketone jpet.aspetjournals.org bodies thereby providing substrates for energy metabolism in peripheral tissues (Issemann and Green, 1990). LXRα, the predominant LXR paralogue in liver (Repa et al., 2000) regulates at ASPET Journals on September 25, 2021 intracellular cholesterol and bile acid metabolism as well as expression of sterol regulatory binding protein (SREBP)-1c, the major lipogenic transcription factor (Schultz et al., 2000; Repa et al., 2000). Activation of LXR is associated with down-regulation of key genes involved in hepatic gluconeogenesis (Stulnig et al., 2002a; Cao et al., 2003; Laffitte et al., 2003). Moreover, nuclear receptors can modulate each other’s gene expression as shown for PPARγ and LXRα (Ide et al., 2003; Seo et al., 2004) pointing to a close relation of their transcriptional regulations and metabolic function. Agonists of PPARγ, PPARα and LXR all decrease blood glucose concentrations in type 2 diabetes patients and/or comparable animal models (Lehmann et al., 1995; Guerre-Millo et al., 2000; Laffitte et al., 2003) by regulating gene expression. In order to address the question whether normalization of blood glucose levels by these nuclear receptor agonists is accompanied by normalized gene expression, we analyzed genome-wide hepatic gene expression profiles of diabetic db/db mice treated with nuclear receptor agonists and their untreated non-diabetic littermates and compared them with 4 JPET Fast Forward. Published on October 31, 2005 as DOI: 10.1124/jpet.105.093831 This article has not been copyedited and formatted. The final version may differ from this version. JPET# 93831 those of untreated db/db mice. By providing a comprehensive overview of drug-induced changes in gene expression in obese diabetic mice our data revealed that reversal of diabetes- associated alterations in hepatic gene expression occurs only to a very limited extent. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 25, 2021 5 JPET Fast Forward. Published on October 31, 2005 as DOI: 10.1124/jpet.105.093831 This article has not been copyedited and formatted. The final version may differ from this version. JPET# 93831 MATERIAL AND METHODS Animals. Male C57BL/KsJ-leprdb/leprdb diabetic (db/db) mice and their nondiabetic littermates (db/+) were purchased from Charles River Laboratories Inc. (Germany) at seven weeks of age and maintained under standard light (12h light/dark) and temperature conditions (23°C). During one week of acclimatization mice were provided with a low-fat standard rodent diet (<3% fat; N1324, Altromin, Germany) and water ad libitum. Downloaded from Treatment. For the experiment, the low-fat standard diet was either mixed with vehicle alone (ethanol; untreated) or supplemented with 0.005% (w/w) PPARγ agonist Rosiglitazone (Rosi; jpet.aspetjournals.org Avandia, Smithkline Beecham, PLC, Middlesex, U.K.; corresponding to approximately 10 mg/kg/day; Hori et al., 2002), 0.02% PPARα agonist Wy-14.643 ([4-chloro-6-(2,3-xylidino)- 2-pyrimidinylthioacetic acid; Wy; Sigma-Aldrich, St Louis, MO, U.S.A.; corresponding to at ASPET Journals on September 25, 2021 approximately 30 mg/kg/day; Edvardsson et al., 1999) or 0.025% of the synthetic LXR agonist T0901317 (N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy- 1(trifluoromethyl)-ethyl]phenyl]-benzenesulfonamide; T09; generously provided by Amgen Inc., formerly Tularik Inc.; corresponding to approximately 40 mg/kg/day; Stulnig et al. , 2002a) followed by extensive evaporation of ethanol. Four groups of db/db (untreated, Rosi, Wy, T09; n = 5) and one group of db/+ mice (untreated; n = 8) were treated for 10 days. Tissue and blood analyses. Mice were anesthetized with Isoflurane and sacrificed by neck dislocation after cardiac puncture. The liver was immediately cut into small homogenous regions, snap frozen in liquid nitrogen and kept at -80°C until isolation of total RNA. Blood samples were drawn from the tail vein before starting the experimental diets. EDTA plasma separated from cardiac blood was stored in aliquots at -20°C until further analyses. Blood glucose was measured by an automated analyzer (ALCYON 300i, Abbott Laboratories, 6 JPET Fast Forward. Published on October 31, 2005 as DOI: 10.1124/jpet.105.093831 This article has not been copyedited and formatted. The final version may differ from this version. JPET# 93831 Illinois, U.S.A.) at the beginning and the end of the experiment. Plasma cholesterol and triglycerides were determined by the Alcyon 300i analyzer (Abbott Laboratories). Serum concentrations of non-esterified fatty acids were measured with the Wako FFA-kit (Wako chemicals,